i
of Enzymes In a previous report, Howard Weetall reviewed the chemistry and biochemistry of enzyme immobilization ( I ) . Since his review emphasized the chemical methods for immobilization, comparisons of the properties of immobilized and free enzymes with respect to pH profiles, thermal stability, and Michaelis-Menten constants, we will assume familiarity with these topics. This report is intended to be an overview of the spectrum of analytical applications for immobilized enzymes in biochemical, environmental, and industrial analysis. The principles of immobilized enzyme electrodes and enzyme reactors will also be discussed. The utility of enzymes as analytical reagents has been well documented (2, 3 ) . Previously, the instability, expense, scarcity, and general lack of familiarity with these biochemicals have discouraged analytical chemists from using them. In the past decade advances in the isolation and purification of proteins have increased the availability of many enzymes. Over 1000 enzymes have been isolated and characterized, thereby providing the basis for selective reactions for determining substrates ranging from simple inorganic species such as nitrate and phosphate ions to macromolecules (3, 4 ) . This indicates the analytical potential inherent in enzyme technology. With the advent of a variety of successful immobilization methods, it appears that enzymes are destined to become routine laboratory tools. The more immediately obvious advantages of insolubilizing an enzyme are its easy introduction into and separation from a reaction mixture and, more importantly, its reuse. This conPresent address, Department of Clinical Pathology, University of Oregon Medical School, Portland, Ore. 544A
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
Larry D. Bowers’ and Peter W. Carr
Report
Department of Chemistry University of Georgia Athens, Ga. 30602
veys a number of advantages. For example, the same immobilized enzyme can be used to catalyze a reaction in a large number of samples and still be recovered. Analytically, this is import a n t since i t enables the analytical chemist to use large amounts of enzyme t o achieve rapid equilibrium type assays a t low cost. Another feature which often accompanies the use of immobilized enzymes is increased p H and/or temperature stability, thereby permitting the use of the enzyme under adverse conditions ( I ). These advantages, combined with the wide variety of enzymes available and the inherent selectivity of enzyme processes, make these materials an important addition to the analytical and clinical laboratory.
Methods for Preparing Immobilized Enzymes Immobilized enzymes may be classified by the method used to prepare them. Four distinct approaches which have been extensively studied are summarized in Table I along with their analytically important features. T h e four techniques are: adsorption onto an insoluble carrier (5-7), covalent crosslinking of the enzyme to itself or a second type of protein, entrapment within a gel matrix ( 8 ) ,and covalent attachment to an insoluble carrier such as glass, cellulose, dextran, or ion-exchange resin. The reader is referred to one of the many excellent recent reviews ( 1 , 9 , 1 0 )or monographs (11-19). Analytical Applications Although the analytical uses of immobilized enzymes have been reviewed elsewhere ( I , 10, 14, 15),these compilations have been somewhat limited in scope. For the purposes of this report, analyses based on immo-
bilized enzymes are arbitrarily divided into three categories: solid-state fluorimetric assays, “enzyme electrode” type devices wherein an artificial enzyme membrane is fixed directly to t h e transducer, and the immobilized enzyme reactor approach with subsequent detection by any convenient method such as colorimetry or amperometry. We will attempt to review the more recent advances in the application of immobilized enzymes to.analytical chemistry with particular emphasis on clinical and biochemical measurements. Some important analytical characteristics of these systems are given in Table 11. Solid Surface Fluorescence. This methodology is essentially an adaptation of solution fluorescence which is carried out on a semisolid surface. The enzymes necessary to catalyze the formation of a fluorescent material along with all other required reagents are lyophilized onto a silicone rubber pad. After reconstitution the analyte solution is placed on the pad and allowed to diffuse into the gel. An interesting example is found in the analysis of serum urea in which urease, glutamate dehydrogenase, and NADH are entrapped in the pad (21). Upon addition of serum, the hydrolysis of urea is coupled to the removal of NADH by the reactions:
+ H + + 2Hz0 urease e 2NH4+ + HC03a-ketoglutarate + NH4+ Urea
+NADH
glutamate
e
(1)
NAD+
dehydrogenase
+ glutamate + HzO
(2)
Quantitation is accomplished by monitoring the change in fluorescence as a function of time. A very similar system for the determination of serum
glucose uses the hexokinase/glucose6-phosphate dehydrogenase/NADP system (22). Solid-state fluorescence has also been used to measure the activity of creatine kinase (23).These methods have not generated a great deal of interest as yet. By and large, the systems developed have been for the detection of enzymes rather than substrates and thus have not generally involved the use of immobilized enzymes. The advantages of the technique include small sample volumes, ease of use, and increased reagent stability. The disadvantages include the necessity for dissolution of the reagent on the pad, relatively high levels of background fluorescence, and a somewhat involved pad preparation procedure.
Transducer-Bound Immobilized Enzymes. Potentiometric. T h e concept of combining the selectivity of an enzyme-catalyzed reaction with the convenience and sensitivity of electrochemical methods is generally attributed to Clark and Lyons (24).The operation of artificial membrane enzyme probes is based on the diffusion-controlled movement of substrate through a thin layer of catalyst. The substrate reacts to form a product species which is detected a t the sensor surface. Thus far, the reactants or products have been quantitated by potentiometry and amperometry. A schematic representation of these devices is shown in Figure l. Enzyme electrodes have flourished due to the number of important enzymes producing electroactive species (25) and the availability of compatible ion-selective electrodes. Although the basic operating concept is the same for both amperometric and potentiometric enzyme probes, there are substantial differences in behavior due to the electrochemical con-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976 * 5 4 5 A
ZEISS
h
Table 1. Comparison of Techniques for Immobilizing Enzymes Method
Limitations
Advantages
Adsorption
Chemically simple; can give high initial yield: widely applicable Can make membranes
Proteincross- linking Entrapment Chemically simple: high initial yield; can make membranes; widely applicable; can be lyophilized Very flexible approach; can achieve Covalent bonding good flow properties (glass support); no leakage of protein
Rigid control of conditions to prevent desorption Poor flow properties; low reactivity for high MW materials Poor flow properties: low reactivity for high MW materials: will leach protein slowly Low initial yield of enzyme activity
Table II. Comparison of Immobilized Enzyme Analytical Systems TYW
Advantages
PROBE Potentlometrlc
Amperometrlc
REACTORS Column
Tubular
Simplicity of operation; easy to make: uses small amounts of enzyme
Simplicity of operation; easy to make; wider linear range: more enzymes produce or consume 0 2 or H202; uses little enzyme Wide variety of enzymes and. detectors; complete conversion: high throughput Low pressure drop; completely compatible with flow analysis; wide variety of enzymes: high throughput
sumption of the product a t the sensor surface of the amperometric devices. Elegant mathematical treatments of mass-transport-coupled enzyme reactions in membranes have been developed (26-29). Rlaedel and coLvorkei-5 (29)have derived equations Lvhich define the steady-state concentration of both the substrate and product for p(itentiometric sensors. Their model includes the effect of film diffusion. i.e.. external mass transport and the selective phase-partitioning of the s u b strate between the external solution and the membrane phase ( 2 9 ~IVhen . such complicating factors are disregarded. one ohtain; the follo\ving equationsfor the ccincentratic~nof product (P) at the sensor surface: =
IPl..ell..,,,
Limitations
[Pll,,,lk
Slow response; memory effects; relatively few enzymes compatible with operation ("3, Con): sensitive to inhibitors and activators; restricted linear range; incomplete conversion Slow response, memory effects: electroactive interferences; sensitivity to materials which consume H202; incomplete conversion Not economic of enzyme; may have high pressure drop; more elaborate system: some solid supports (porous glass) expensive May be very long for some enzymes; may have long start-up time
where:
x,n;],
The terms f, and D,repre.qent the enzyme concentration per unit volume of gel imoljcc-s).t h r thickness of the enzyme memhrane (mi) . and the diffusion coefficients of the substrate and product. respectivc,Iy (cms/'s). in the membrane phase. The quantity n is the appropriate stoichiometry coefficient for conversi:in of the substrate to product. e.g.. n = 2 for urea when NH or NH4+ is tietected. As is the case a.ith all enz:;matic rat e analyses, the tneay i i r eti p a r a meter ([f?],,,,.,,,) become> independent of the substrate at very high c.oncrntration (see Equation 3 and Figure 2 : . A t high levels of' enzyme ac,tivity i TIIi,,?I or with thick membranes ix).P:qtiation 4 simplifies to:
We have dropped the term [ P ] b u i k to emphasize the linear relationship between the product concentration a t the sensor surface and the bulk substrate concentration. A sensor such as a n ion-selective electrode will be responsive to any material which is converted to the product, but will also be sensitive to the bulk concentration of product. The advantages of the potentiometric membrane probe are its simplicity, reliability, and low cost. The limitations of the technique are the result of a number of fundamental factors. First, an enzymatically generated species is required for which an ionselective electrode exists. A second problem is the somewhat limited selectivity of the electrodes which may result in susceptibility to ionic interferences. An excellent example of this problem and its solution is evident in the evolution of several generations of urea electrodes. In the first paper on enzyme-ion-selective electrodes, Guilbault and Montalvo used a layer of polyacrylamide gel-entrapped urease over the surface of a glass cation-selective electrode to detect ammonium ions (30). This particular electrode exhibited response to sodium and potassium ions as well as to the ammonium ion produced by the hydrolysis of urea, thus necessitating a n ion-exchange pretreatment of biological fluid before analysis. To overcome this difficulty, Guilbault et al. showed t h a t a more selective ammonium ion-selective electrode (nonactin-silicone rubber membrane) could be used, provided t h a t the sample’s potassium level was adjusted appropriately ( 3 1 ) .For greater specificity, Anfalt et al. found t h a t urease could be covalently bound t o the gaseous diffusion membrane of an ammonia-selective electrode and the free ammonia quantitated ( 3 2 ) .A similar approach using an entrapped enzyme layer and a Copselective electrode was attempted (33, 3 4 ) . Both of these gas diffusion electrodes are slow ( 5 min a t M), and the membrane will clog in biological matrices. The most recent approach to the analysis of urea is the air gap electrode introduced by Hansen and Ruzicka in which the p H electrode and its electrolyte film are separated from the analyte solution by a thin air layer ( 3 5 ) . T h e ammonia produced in the reaction of the urea with immobilized urease beads is trapped in a gas-tight system and diffuses into an electrolyte solution. The change in the p H of this electrolyte is then used to quantitate the substrate (36).Although this is not strictly an “enzyme electrode”, but rather a microflow system, it does provide the advantage that the electrode membrane is not in contact with the solution and therefore does not
hOt OVERHANG PROTECTS INNARDS INFINITE AT WITHIN RANGE
1
PC-351HAS 35 INZ
BIG KNOBS EASIER T O TURN,READ
1
950’F IN 7 TO 9 MIN
CORNING
CORNING”
hotwmm CIRCLE 3 4
ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
547A
Figure 1. Schematic diagram of electrochemical enzyme probes. A : Potentiometric sensor; E3: ainperometric sensor
To
Figure 2. Schematic diagram of immobilized enzyme-chemiluminescent analyzer for serum gluccise (62) 548A
ANALYTICAL CHEMISTRY. VOL 48, NO 7, JUNE 1976
become clogged. Recent work by Blaedel and Kissel in which a urease membrane was joined to a n anion-exchange membrane indicates that the selectivit y problem of the cation NHA+ selective electrode might be avoided by using the electrostatic exclusion phenomena of an ion-exchange material ( 9 4 ) .Their work demonstrates the powerful advantages inher'ent in the use of membrane technology for simultaneous separation and detection. A third problem which has been alluded to above is the slow response time of the device. By its nature, the sensor requires 'diffusion of the substrate and product into and out of the enzyme layer. Such diffusism may be relatively slow, Iparticularlg over the distances normally encountered in artificial membranes (0.01 crn). A second problem propagated by this same process is sample carry-over or membrane memory effects which reduce the sample throughput even further. Des pi te these li imi t a t ions, po ten tio metric enzyme electrodes are very much in vogue as illustrated by the multitude of such systems given in Table 111. Amperometric. Although the a m perometric electrode was the first type of enzyme probe reported in the literature ( 3 7 ) ,it has only recently received substantial attention. The amperometric probe is based on the proportionality between the observed current and the amount of electroactive species generated in the membrane phase. The oxygen cr hydrogen peroxide consumed or produced by certain enzyme reactions in a membrane provides ,a convenient analytical handle for amperometric detectors. Although the two types of electrochemical transducers are c,perationally similar. their performance may be significantly different. One of the major distinctions between the potentiometric and amperometric proties is the wider linear range of the amperometric probe, i.e., this sensor can under some circumstances provide a linear response at concentrations much greater than the Michaelis constant of the substrate-enzyme system. This phenomenon can be observed in the potentiometric ( 7 1 ) and arnperometric (76) glucose prclbes which are designed around the use of glucose oxidase. The linear range of t:ne amperometric probe not only exceeds that of the potentiome1:ric probe by an order of magnitude, but also exceeds the K , of the enzyme. behavior which is not generally obserT:ed Lvith the potentiometric device. This apparent discrepancy has been explained hy the fact that the amperometric electrode removes the reaction product from solution, thereby altering its concentration profile in the
SARGENT-WELCH
in the Sargent-Welch tradition of fine recorders,..
-
Anaheim
Sargent-Welch Scientific Company 0 Birmingham 0 Chicago 0 Cincinnati
0 0
7300 North Linder Avenue Cleveland 0 Dallas 0 Denver
0 0
Skokie, Illinois 60076 Detroit 0 Springfield, N.J.
0 0
(312) 677-0600 Toronto
0
Montreal
CIRCLE '192 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
549A
Table 111. Potentiometric Enzyme Probes Anaiylo
Enzyme
Trmsducar
L-amino acids L-amino acid oxidase D-amino acids D-amino acid oxidase Amygdalin (?-glucosidase
Ref
C-nt./F.wtt.
Cation selective
Na+,K+ interference; precision 2.5%
66
Cation selective
ion-exchange pretreatment necessary
67
CN’ ion selective
Very slow response (30 min for M); enzyme paste used Formation of urea by arginase followed with urea enzyme electrode
68,69
Arginase
Urease
Asparaglne Glucose
Asparaginase
Glutamine Penicillin
Glutaminase Penicillhase
Cation selective pH electrode
L-tyrosine
COPselectlve
Urea Urea
L-tyrosine decarboxylase Urease Urease
Urea
Urease
Urea
Urease
Cation selective n solution to react with
70 67 71
hydrogen peroxkle
An important feature here is the strong inverse dependence on the membrane thickness. At sufficiently high substrate concentration, regardless of the amount of enzyme, the current becomes independent of the substrate concentration. The calculations of Me11 and Maloy were the first to yield theoretical estimates of the response time of an enzyme electrode. 550A
72 73,74 33 30 31
s in serum give unstable readings;slow
ioxide selective Ammonia selective
enzyme layer. Me11 and Maloy, using numerical techniques to solve the boundary value problem, have shown that the current may be limited by either the rate of the enzyme reaction or by the rate of diffusion (38).If the catalytic rate is limiting and the bulk concentration is low, i.e., [SI > K,, the extent of the reaction is simply proportional to the amount of time spent in the reactor:
[S],F = v
(1 - € ) L
(12)
U O
Unfortunately, the situation is kinetically much more complex than that presented above. Two categories of problems are inherent in the use of immobilized enzyme reactors. The first class, enzymatic problems, includes microenvironmental factors, product and substrate inhibition, and thermal inactivation. These have been discussed a t length elsewhere (48,491. A second set of problems is present in the mass transfer processes which restrict the design of all chemical reactors. Analogous to the situation which 554A
Glucose oxldase
Comments/r.urHs
Depletion of O2at Clark oxygen electrode
Ref
37
Response time
